Epithelial-mesenchymal transition (EMT) contributes to cell invasion and
metastasis during the progression of epithelial
cancers. Though preclinical evidence suggests a role for
histamine H4 receptor (H4R) in
breast cancer growth, its function in the EMT is less known. In this study we proposed to investigate the effects of H4R
ligands on EMT and mammosphere formation as a surrogate assay for cancer stem cells in
breast cancer cells with different invasive phenotype. We also investigated the participation of Src and TGF-β signaling in these events.
Breast cancer cells were treated with the H4R agonists
Clobenpropit,
VUF8430 and
JNJ28610244 and the H4R antagonist
JNJ7777120. Immunodetection studies showed cytoplasmic
E-cadherin, cytoplasmic and nuclear
beta-catenin, nuclear Slug and an increase in
vimentin and α-smooth muscle actin expression. There was also an enhancement in cell migration and invasion assessed by transwell units. All these effects were prevented by
JNJ7777120. Moreover, H4R agonists induced an increase in phospho-Src levels detected by Western blot. Results revealed the involvement of phospho-Src in EMT events. Upon treatment with H4R agonists there was an increase in phospho-ERK1/2 and TGF-β1 levels by Western blot, in Smad2/3 positive nuclei by indirect immunofluorescence, and in
tumor spheres formation by the mammosphere assay. Notably, the selective TGF-β1
kinase/activin receptor-like kinase inhibitor
A83-01 blocked these effects. Moreover, cells derived from mammospheres exhibited higher Slug expression and enhanced migratory behavior. Collectively, findings support the interaction between H4R and TGF-β receptor signaling in the enhancement of EMT features and mammosphere formation and point out intracellular TGF-β1 as a potential mediator of these events.